The transport of L-malic acid across the plasma membrane and its degradation in microorganisms is of considerable interest in many fields, particularly those involving fermentation by yeasts. L-malic acid may be used as a sole carbon and energy source by the yeasts Candida sphaerica (Corte-Real et al., 1989), Hansenula anomala (Corte-Real and Leao, 1990) and Candida utilis (Cassio and Leao, 1993). The dissociated form of malate is transported across the plasma membrane by proton symports which are inducible and subjected to glucose repression. However, in Zygosaccharaomyces bailii (Rodriquez and Thornton, 1990) and Schizosaccharomyces pombe (S. pombe) (Sousa et al., 1992), L-malic acid can only be metabolized in the presence of an assimilable carbon source (Osothsilp and Subden, 1986). L-malic acid is actively transported in the dissociated form whereas the undissociated acid enters the cell via simple diffusion (Baranowski and Radler, 1984; Osothsilp and Subden, 1986; Sousa et al., 1992). Competitive inhibition of initial uptake rates of L-malic acid by succinic acid, D-malic acid, faumaric acid, oxaloacetic acid, .alpha.-ketoglutaric acid, maleic acid and malonic acid strongly suggests that these acids are transported by the same carrier in S. pombe (Sousa et al, 1992).
Malic acid degradation is of particular interest to wineries. Wine yeast strains of Saccharomyces cerevisiae (S. cerevisiae) cannot metabolize malate in grape must efficiently and changes in the total acidity of the wine during vinification are therefore insignificant (Gao, 1995). Production of well-balanced wines requires the controlled reduction of excess malic acid, particularly in the colder viticultural regions of the world.
Chemical deacidification has been used to reduce the total acidity of wine. Chemical deacidification is typically carried out by (a) amelioration--which is essentially dilution of the malic acid with sugar water; (b) precipitation--the addition of calcium, potassium or other cations to produce an insoluble salt; or (c) masking--adding grape juice or sucrose to the finished wine to mask the sour taste of malic acid. All these methods result in residual malate which can support malolactic fermentation by contaminating bacteria unless treated with elevated doses of sulfites.
Malolactic fermentation methods for malic acid degradation rely on the conversion of L-malic acid to L-lactic acid and CO.sub.2 by malolactic bacteria, for example, species of Leuconostoc, Lactobacillus, and Pediococcus. The malolactic bacteria may be found on grapes which become part of the winery microflora, or commercially available frozen or freeze-dried cultures of the bacteria may be introduced into the wine. Malolactic fermentation methods have a number of disadvantages; for example, the malolactic bacteria ferment terpenes which change the character of the wine. Control of malolactic fermentations is often difficult resulting in incomplete malolactic fermentation and subsequent bottle fermentations. Bacterial growth is also usually accompanied by the production of carbon dioxide which may result in "fizzy" wine.
Yeast strains which can degrade L-malic acid have also been used in wine fermentations. Fermentations using the fission yeast S. pombe which completely degrades malate to ethanol through a malo-ethanolic fermentation have been attempted. Thornton (U.S. Pat. No. 4,830,968) describes a method involving inoculating grape juice with a strain of Saccharomyces malidevorans which is capable of some degradation of L-malic acid under wine making conditions. However, these yeast strains (i.e. Schizosaccharomyces pombe and Saccharomyces malidevorans) are not desirable in wine making since off-flavours are produced. High density cell suspensions of several yeasts, including S. cerevisiae have also been used to try to increase the rate at which L-malate is degraded during fermentation (Gao, 1995).
Attempts have been made to hybridize wine yeasts with malate-metabolizing yeast strains. Protoplast fusion (Carrau et al., 1982; Svoboda, 1980, U.S. Pat. No. 5,330,774 to Carrau et al.), transformation (Lautensach and Subden, 1984; Williams et al., 1984), and other means (Fernandez, 1967; Goto et al., 1978; Kuczynski and Radler, 1982) have not been successful.
Metabolic engineering of S. cerevisiae strains to carry out alcoholic fermentation and malolactic or malo-ethanolic fermentation simultaneously has been explored. The malolactic gene (mleS) from Lactobacillus delbrueckii (Williams et al., 1984) and Lactococcus lactis (Ansanay et al., 1993, Denayrolles et al., 1994) have been cloned, characterized and several attempts have been made to introduce and express this gene in S. cerevisiae. However, recombinant strains of S. cerevisiae expressing the mleS gene were unable to degrade malate effectively to L-lactate (Williams et al., 1984; Ansanay et al., 1993, Denayrolles et al., 1995).